Sandbox 47

Bovine Pancreatic Trypsin Inhibitor (BPTI) Mutant with Altered Binding Loop Sequence  BPTI is a single chain polypeptide with 58 amino acids which inhibits the serine protease Trypsin. It's secondary structure includes one beta sheet and two alpha helices. The beta sheet consists of two antiparallel strands (Ile18 to Asn 24 and Leu 29 to Tyr 35) with a beta hairpin consisting of four residues (Ala 25, Lys 26, Ala 27, Gly 28). Alpha helix H1 is 4 residues (Asp 3 to Cys 6), and H2 is 8 residues (Ala 48 to Cys 55). There are three intrachain disulfide bonds in this polypeptide, one between Cys 5 and Cys 55, one between Cys 14 and Cys 38 , and one between Cys 30 and Cys 51. These are important for BPTI's stability and its ability to withstand the harsh conditions of the digestive system. This protein has a hydrophobic core (gray) with polar and charged residues (purple) extending mostly on the surface. The N-terminus and C-terminus are in contact through a salt bridge. BPTI interacts with 98 water molecules as well as 4 sulfate anion ligands. Ligand 61 binds the protein through hydrophobic interactions between Glu 7, Lys 7, and Phe 41, as well as through 3 hydrogen bonds to Arg 42. Three water molecules are also interact with this ligand. Ligand 62 has hydrophobic interaction with Ala 40, two hydrogen bonds with Arg 20, and one hydrogen bond with Tyr 35. Ligand <scene name='Sandbox_47/63/1'>63 has hydrophobic interaction with Pro 2 and one hydrogen bond with Arg 1. Ligand <scene name='Sandbox_47/Ligand_64/1'>64 has hydrophobic interaction with Cys 14.

BPTI has both a <scene name='Sandbox_47/Primary_loop/1'>primary and a <scene name='Sandbox_47/Secondary_loop/1'>secondary loop involved in the binding and inhibition of Trypsin. This particular mutant of BPTI contains several residue substitutions within these loop, including a substitution at <scene name='Sandbox_47/Lys_mutation/1'>residue 15, which is is normally Lys but has been replaced by Arg. This basic Lys normally binds to the active site of Trypsin and is therefore important to the inhibition of the protease. This mutant also has <scene name='Sandbox_47/Thr11_sub/1'>Thr11Ala, <scene name='Sandbox_47/Sub/1'>Pro13Ala , and <scene name='Sandbox_47/Met_52_sub/1'>Met52Leu substitutions. Altogether these substitutions reduce BPTI's association constant by approximately 30 times (Czapinska et al,1999).

Resources: Czapinska, H., Otlewski, J., Krzywda, S., Sheldrick, G.M, Jaskilski, M. (1999) "High-resolution structure of bovine trypsin inhibitor with altered binding loop sequence." J. Mol. Biol. 295:1237